SBB09Ntbk-Jennifer Brophy

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Jennifer Brophy 00:03, 9 February 2009 (EST)

If possible, make two circularly permutated ompG proteins. One with the backbone opening in loop 2 (aa 59-60) and one in loop 6 (somewhere within aa 220-231) where the residues of the mature protein are not visible in electron density maps, presumably because they are disordered.

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Jennifer Brophy 11:59, 9 February 2009 (EST)

Created Construction files for CPG_L2 and CPG_L6. Entered oligos in oligo log

BLAST oligos, enter parts in parts log, look into Nterm and Cterm linker sequences.

Jennifer Brophy 13:19, 18 February 2009 (EST)

Reconstitute oligos. 100uM by adding 10X nmol.

PCR of M10006 (<N.CPG_L2!, regular part, 211bp), A-M10007 (371bp), B-M10007 (343bp), A-M10009 (548bp), B-M10009 (175bp), M10010 (<C.CPG_L6>, regular part, 202bp).

zymo clean-up, PCR A+B for M1007 and M1009.

Jennifer Brophy 21:02, 19 February 2009 (EST)

Did zymo clean up of PCR products (small fragment and regular clean up where appropriate). Diluted some clean ups in to much water so i either redid the PCR or added more template for the A+B reactions. Did PCR A+B of M10007 (694bp) and M10009 (703bp). BUT THESE DIDNT WORK BECAUSE I FORGOT TO DO GEL PURIFICATION TO GET RID OF TEMPLATE DNA.

Jennifer Brophy 18:57, 20 February 2009 (EST)

Did gel purification of M10006,M10007A, M10007B, M10009A (no 9B because i had messed up the zymo clean up yesterday), and M100010. Failure to remove the genomic DNA via gel purification yesterday rendered the PCR of M10007 and M10009 useless.

Gel=ladder, M10006 (211bp), M10007A (371bp), M10007B (341bp), M10009A (548bp), M10010 (202bp).
Re-did PCR of M10009B.

gel purify M10009B. do next round (A+B) PCR. gel purify? start SOEing

Jennifer Brophy 13:07, 23 February 2009 (EST)

Zymo small fragment clean up of M10009B. Gel purify M10009B.

Gel= ladder, M10009B (175bp).
Do A+B PCR to get M10007 (694bp) and M10009 (703bp).

gel= ladder, M10007, M10009. Careful gel purification of M10007, less of a product, mixture of oligos?
Set up PCR of M10008A/B and M10011A/B.

Jennifer Brophy 12:23, 24 February 2009 (EST)

Gel purify M10008A (209bp), M10008B (701bp), M10011A (701bp), M10011B (209bp).

Gel= ladder, M10008A, M10008B, M10011A, M10011B.
Set up PCR for M10008 and M10011.

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Jennifer Brophy 13:27, 25 February 2009 (EST)

Gel purify M10008 (892 bp) and M10011 (892bp).

Gel= ladder, M10008, M10011.
Did Eco/Bam digest and ligation into pBca9495KC. Plated on KC plates.

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Jennifer Brophy 13:29, 27 February 2009 (EST)

Innoculate two tubes of M10011.
Re-do ligation of M10008 because no colonies grew. Redo plating.

pick M10008 colonies. 
miniprep colonies. Do restriction mapping: (parts >250 bp= EcoRI/BamHI) 
 6.5uL water
 1 uL NEB2
 2 uL plasmid
 0.5 uL EcoRI
 0.5 uL BamHI or XhoI
30 min at 37C 
run gel
turn in for sequencing

Jennifer Brophy 01:18, 1 March 2009 (EST)

Picked M10008 colonies. Plate looked very good. Many colonies grew.

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Jennifer Brophy 17:08, 1 March 2009 (EST)

Did miniprep of M10008A/B (the two colonies), and M10011A/B.

Gel=ladder, M10008A, M10008B, M10011A, M10011B
Restriction map looks good.

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Jennifer Bophy 14:36, 2 March 2009 (EST)

Sent in M10008 and M10011 for sequencing as sb0001, sb0002, sb0003, sb0004.

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Jennifer Bophy 14:36, 4 March 2009 (EST)

Analyzed sequencing results of sb0001 (M10008 ad 3 silent point mutations, the part number was changed to M10086) and sb0002 (M10011 was perfect).
Did gateway transfer of Bjb2 {<cpx!} into pBca9495KC.

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Jennifer Bophy 13:41, 6 March 2009 (EST)

Miniprep of M10063A and M10063B (two clones of M10063).
Did restriction digest of M10063.
Sent in M10063 for sequencing as sbb052 and sbb053.

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Jennifer Bophy 13:23, 11 March 2009 (EDT)

Analyzed the sequence of sbb052: perfect result for M10063.

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Jennifer Bophy 14:41, 20 April 2009 (EDT)

Started the Cell-cell adhesion assay. Transformed pBjb1600-Bca1144 (RFP) into DH10B cells. Plated on Spec plates. These cells will eventually be co-transformed with the IILK and AG4 plasmids to test cell density at the end of our experiments.
We are also doing a round of clones without the RFP plasmids, but we did not transform any DH10B cells with plasmids because we are hoping that other groups will have extra colonies.

Pick colonies (3 of each), grow to saturation, dilute, ect.

Jennifer Bophy 15:47, 21 April 2009 (EDT)

Picked colonies into 24 well plate with 3mL of LB. First two columns (A1-D1 and A2-D2) are pBca9495CA-(M10218-M10225), the next two columns are pBca9495CA-(M10210-M10217). A5 is pBca9495CA-Bca1363 and B5 is pBca1600-Bca1144 (RFP-plasmid).

Do dilutions. Re-transform RFP cells with corresponding plasmids.

Jennifer Bophy 14:24, 22 April 2009 (EDT)

Dilute cells into 96-well plate (to go into the shaker) and V-bottom plate (to go into the incubator). In the 96 well plate put in 1 mL of LB (with or without arabinose) and 100uL of saturated cell culture. In the V-bottom plate, put in 300uL of LB (with or without arabinose) and 30uL of saturated cell culture.
In V bottom plate:

A1-8=IILK with arabinose
B1-8=IILK without arabinose
C1-8=AG4 with arabinose
D1-8=AG4 without arabinose
E1=1363 with arabinose
E2=1363 without arabinose

In 96 well plate:

A1-8=IILK without arabinose
B1-8=IILK with arabinose
C1-8=AG4 without arabinose
D1-8=AG4 with arabinose
E1= 1363 without arabinose
E2= 1363 with arabinose

The RFP cells were also transformed with our plasmids of interest.

OD measurements, look at V-bottom plate, pick colonies.

Jennifer Bophy 15:21, 23 April 2009 (EDT)

Did OD measurements of colonies grown in 96 well plate (see David's page).
V-bottom plate was difficult to ascertain results from. A4 and A7 do not have pellets of ecoli at the bottom, and A8's pellet is very small. Everything else looks the same (big pellet).

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Jennifer Bophy 19:09, 27 April 2009 (EDT)

Re-transformed man19-man26.

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Jennifer Bophy 19:09, 28 April 2009 (EDT)

Picked colonies ( man19-26 and Bca1363) into 3mL of LB in 24 well plate.

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Jennifer Bophy 19:09, 29 April 2009 (EDT)

Diluted colonies (man 19-26 and Bca1363), 100uL into 1mL of media. Did two replicates of each colony, one with arabinose and one without.

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Jennifer Bophy 19:09, 30 April 2009 (EDT)

Tried to take OD measurements with sodium hydroxide add as control to reverse flocculation. Realized that sodium hydroxide kills cells and does not disrupt leucine zipper. Tried to find trypsin without dye. Couldnt find it.

Need to repeat experiment with protein kinase A.

Jennifer Bophy 13:20, 4 May 2009 (EDT)

Start cell-cell adhesion assay over again. Pick colonies of man19-26 and 1363.

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Jennifer Bophy 19:52, 5 May 2009 (EDT)

Diluted colonies (2 replicates each) with and without arabinose, as stated in "cell-cell adhesion" protocol.

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Jennifer Bophy 19:52, 6 May 2009 (EDT)

Took OD measurements of all samples. Added 1uL of proteinase K to each sample. Incubated for 1 hr at 37.
Took new OD measurements. We were hoping that the proteinase would chew up all of the exposed outer membrane proteins so that the leucine zippers would no longer be functional. This way we could normalize OD measurements of flocculating colonies with OD measurements from solutions of individual cells.
Unfortunately the proteinase K did not work, we saw little to no change in OD measurements or visible clumping (see Sam Ng's notebook for raw data).
This meant that we needed to revert back to our original control (of M10011-M10017, these have AG4 as the displayer peptides).

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Jennifer Bophy 19:52, 7 May 2009 (EDT)

Picked new colonies (two each) of M10011-M10026 and 1363 for the last run of the cell-cell adhesion assay.

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Jennifer Bophy 20:01, 8 May 2009 (EDT)

Diluted two replicates of each clone into 96 well plate. One replicate with, one without arabinose.

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Jennifer Bophy 20:03, 9 May 2009 (EDT)

Took OD measurements of every sample. Saw some clumping, OD measurements coincided with clumping observations. Data will be typed up tomorrow for Cell-cell adhesion write up in the registry.

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